Escherichia coli is the best known cellular organism, but many of its genes remain to be identified. The experiments proposed in this revised application will identify previously unknown genes essential for E. coli viability, and also regions of the bacterial chromosome that are free of essential genes, using transposon-based reverse genetic strategies. We will, in particular, identify essential genes that: (i) are expressed only at low levels, (ii) are needed for growth only aerobically, only anaerobically, or only at particular temperatures; and (iii) that encode membrane proteins. Some of these genes will be sequenced, and their protein products characterized biochemically and genetically. Their roles in bacterial growth will be deduced using conditional (heat- and cold- sensitive) mutations. These studies will characterize genes whose products could serve as good targets for the rational design of new drugs of importance in microbial infections, and should provide significant new insights into fundamental principles of bacterial growth.